Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of the pre-implantation blastocyst. ESCs provide a powerful platform for elucidating gene function and creating disease models. So far, the application of ESC-based technologies has been limited to mice and rats, because germline competent ESCs have not yet been established from non-rodent species. Our previous work established that efficient self- renewal of mouse and rat ESCs could be achieved by dual inhibition of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK). GSK3 has two paralogous members, GSK3? and GSK?, which share nearly identical kinase domains. Similarly, extracellular signal-regulated kinase (ERK), the only known physiological substrates of MEK, also has two highly homologous paralogs, ERK1 and ERK2. Despite their high levels of homology, GSK3? and GSK3? possess distinct functions in regulating ESC self-renewal; the same is the case for ERK1 and ERK2. Therefore, fine-tuning of GSK3 and ERK signaling becomes critical in achieving optimal ESC self-renewal. By taking advantage of the chemical-genetic approach and the ESC platform, we propose to investigate the molecular mechanisms underlying the distinct functions of individual GSK3 and ERK paralogs in ESC self-renewal. In Aim 1, we will identify and characterize GSK3 paralog- specific substrates and downstream targets. We will also determine the amino acid variances responsible for the distinct functions of GSK3? and GSK3?. In Aim 2, we will identify and characterize ERK paralog-specific substrates in the cytoplasm and nucleus and investigate how subcellular localization of ERK1 and ERK2 affects their functions in ESCs. In Aim 3, we will fine-tune GSK3 and ERK signaling in mouse and human nave ESCs to achieve optimal self-renewal effect. Success with these three aims will not only provide insights into the molecular basis of ESC self-renewal, but will also have far-reaching implications for our deep understanding of pathological conditions caused by dysfunction of GSK3 and ERK.